Dataset Information

Nonribosomal Peptide Extension by a Peptide Amino-Acyl tRNA Ligase.

ABSTRACT: The catalytic use of a small peptide scaffold for the biosynthesis of amino acid-derived natural products is a recently discovered new biosynthetic strategy. During this process, a peptide-amino acyl tRNA ligase (PEARL) adds amino acids to the C-terminus of a small peptide scaffold in an ATP- and tRNA-dependent process. The mechanism of this unusual transformation is currently not known. In this study, we present a detailed biochemical and mechanistic study of TglB (UniProtKB-F3HQJ1), a PEARL that catalyzes the addition of Cys to the C-terminus of the peptide TglA in the biosynthesis of 3-thiaglutamate in the plant pathogen Pseudomonas syringae. TglB recognizes several important residues close to the C-terminus of TglA to perform its activity and is tolerant with respect to the last amino acid of its substrate peptide. The enzyme recognizes the acceptor stem of tRNACys, as micro- and minihelices, truncated versions of full-length tRNACys that contain the acceptor stem, were also accepted. Mutagenesis of conserved residues in TglB identified several key residues for catalysis and did not support the possibility of TglB adopting various ping-pong mechanisms to catalyze the amino acid addition reaction. Using isotopic labeling studies, we demonstrate that ATP is used to directly phosphorylate the C-terminal carboxylate of TglA. Collectively, the data support a general mechanism for the amino acid addition reaction catalyzed by this class of enzyme.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC6927032 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Nonribosomal Peptide Extension by a Peptide Amino-Acyl tRNA Ligase.

Zhang Zhengan Z van der Donk Wilfred A WA

Journal of the American Chemical Society 20191209 50

The catalytic use of a small peptide scaffold for the biosynthesis of amino acid-derived natural products is a recently discovered new biosynthetic strategy. During this process, a peptide-amino acyl tRNA ligase (PEARL) adds amino acids to the C-terminus of a small peptide scaffold in an ATP- and tRNA-dependent process. The mechanism of this unusual transformation is currently not known. In this study, we present a detailed biochemical and mechanistic study of TglB (UniProtKB-F3HQJ1), a PEARL th ...[more]

PMID: 31751505

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Project description:Recent papers have reported dipeptides containing d-amino acids to have novel effects that cannot be observed with ll-dipeptides, and such dipeptides are expected to be novel functional compounds for pharmaceuticals and food additives. Although the functions of d-amino acid-containing dipeptides are gaining more attention, there are few reports on the synthetic enzymes that can accept d-amino acids as substrates, and synthetic methods for d-amino acid-containing dipeptides have not yet been constructed. Previously, we developed a chemoenzymatic system for amide synthesis that comprised enzymatic activation and a subsequent nucleophilic substitution reaction. In this study, we demonstrated the application of the system for d-amino acid-containing-dipeptide synthesis. We chose six adenylation domains as targets according to our newly constructed hypothesis, i.e., an adenylation domain located upstream from the epimerization domain may activate d-amino acid as well as l-amino acid. We successfully synthesized over 40 kinds of d-amino acid-containing dipeptides, including ld-, dl-, and dd-dipeptides, using only two adenylation domains, TycA-A from tyrocidine synthetase and BacB2-A from bacitracin synthetase. Furthermore, this study offered the possibility that the epimerization domain could be a clue to the activity of the adenylation domains toward d-amino acid. This paper provides additional information regarding d-amino acid-containing-dipeptide synthesis through the combination of enzymatic adenylation and chemical nucleophilic reaction, and this system will be a useful tool for dipeptide synthesis.IMPORTANCE Because almost all amino acids in nature are l-amino acids, the functioning of d-amino acids has received little attention. Thus, there is little information available on the activity of enzymes toward d-amino acids or synthetic methods for d-amino acid-containing dipeptides. Recently, d-amino acids and d-amino acid-containing peptides have attracted attention as novel functional compounds, and d-amino acid-activating enzymes and synthetic methods are required for the development of the d-amino acid-containing-peptide industry. This study provides additional knowledge regarding d-amino acid-activating enzymes and proposes a unique synthetic method for d-amino acid-containing peptides, including ld-, dl-, and dd-dipeptides.

Dataset Information

Nonribosomal Peptide Extension by a Peptide Amino-Acyl tRNA Ligase.

Publications

Nonribosomal Peptide Extension by a Peptide Amino-Acyl tRNA Ligase.

Similar Datasets

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